HCV (hepatitis C virus) was first found as a major pathogenic microorganism of non-A and non-B (NANB) type hepatitis by Chiron Company in U.S.A. in 1989 (Choo, Science, 1989, 244:359-362). The development of HCV diagnostic kit facilitated the research on the actual state of HCV infection. In result, it has been reported that 170 million people have been infected with HCV all over the world, and 1.5% of the Korean population are HCV carriers. Once infected with HCV, 85% of carriers pass into chronic state, which is very high ratio, and chronic HCV leads to hepatocirrhosis and liver cancer (Bisceglie, Hepatoloty, 1997, 26: 345), recognizing HCV to be a cause of very threatening disease. However, an effective treatment agent and a vaccine for HCV have not been developed yet.
The states of immunity of both patients recovering from HCV infection and those passing into chronic hepatitis were clinically investigated. While HCV specific cellular immune response was observed in the patients in recovery, the response was not detected in patients switched over chronic hepatitis (Rehermann, J. Virol., 1996, 70:7092; Lechner, J. Immunol., 2000, 30:2479). Especially, CD4+ Th1 immune response has been known to be closely related to the protection and the recovery from HCV infection (Rosen, Hepatology, 2002, 35:190; Sarih, Immunol. lett., 2000, 74:117; Diepolder, J. Mol. Med., 1996, 74:538). According to the research by Pape group, the conditions of patients infected with acute hepatitis are classified into three types; 1) strong CD4+ Th1 immune response is observed in the patients group free from the virus, 2) CD4+ T cell immune response is not observed in the patients group switched over chronic hepatitis, and 3) CD4+ T cell immune response is seen at early stage when the virus is in control, but as the immune response weakens, HCV returns (Gerlach, Gastroenterology, 1999, 117:933). The above studies support the importance of CD4+ Th1 immune response in the control or elimination of HCV. It was also reported from the studies using a chimpanzee, a unique test animal in which HCV infection and replication are allowed, that rather cellular immunity than humoral immunity played an important role in recovery from HCV infection (Cooper, Immunity, 1999, 10:439). Such results suggest that a strong multi-epitope specific Th1 immunity is required for the prevention and the treatment of HCV. Thus, the development of a vaccine for the prevention and the treatment of HCV are now focused on inducing the optimum Th1 cellular immune response.
A subunit protein vaccine was the first HCV preventive vaccine developed by using surface proteins of HCV, envelop 1 and 2 (E1, E2). HCV E1E2 is surface protein of the virus binding to a receptor of a host cell as being infected. If the host cell has a neutralizing antibody against the protein, HCV infection can be prevented. E1E2 subunit vaccine was tried in chimpanzees, precisely, homologous challenge was tried with 10 CID50 of infection dose. As a result, HCV infection was successfully prevented in 5 out of 7 chimpanzees, and even after being infected, the rest 2 chimpanzees did not progress to chronic hepatitis (Choo, Proc. Natl. Acad. Sci. USA, 1994, 91:1294). That was the first report on HCV preventive vaccine. Even though the effect of the vaccine was proved in chimpanzees, the study has limitations as follows. First, small dose of challenging HCV (10 CID50) was inoculated at the peak time of antibody response. Generally, a vaccine has to protect against viral infection by memory response in any circumstances. In the above report, though, antibody response decreased rapidly after the challenging time point. Thus, it is doubtful that the similar protective effect can be achieved when challenge is done after the antibody response decreased. Second, the vaccine was effective against homologous challenge but not heterologous challenge. Considering HCV has at least 6 major genotypes and great numbers of subtypes (Bukh, 1995, Semin Liver Dis 15: 41-63), a vaccine must have preventive effect against heterologous challenge at least with in the same genotype. Third, the protective effect of the vaccine depended on not cellular immune response but antibody response. Recently, along with the reports announcing the importance of cellular immunity for the protective immunity to HCV, reports asserting the limitation of antibody response have been made (Cooper, 1999, Immunity, 10: 439; Esumi, 2002, Vaccine, 20:3095-3103). So, the protein vaccine depending on antibody response alone as a defense mechanism is questionable. Therefore, attempts to induce cellular immunity with DNA vaccine have been made, and as an example, immunity was induced by DNA expressing E2, a surface protein of HCV, resulting in the protective effect against challenge with 100 CID50 of homologous monoclonal HCV (Forns X., Hepatology, 2000, 32(3): 618-25). It was meaningful as the first report on the preventive vaccine using DNA inducing cellular immunity in chimpanzees, but still had problems, too. First, the challenge was performed at the peak time of immune response. Second, monoclonal HCV challenging inoculum was used for the challenge. Like HCV or HIV (human immunodeficiency virus), the virus that uses error-prone RNA dependent RNA polymerase for replication is characterized by producing numbers of quasispecies. Such variety of quasispecies plays an important role for HCV to establish chronic infection (Farci P, 2000, Science, 288:339). According to a recent study, HCV can be produced by intrahepatic injection of HCV RNA into chimpanzee (Kolykhalov, 1997, Science, 277: 570-4). The recovered HCV was infectious and used in the study by Forns et al. Since the monoclonal HCV cannot provide a variety of quasispecies which exist in reality, whether the protective effect is still the same when a real infectious HCV attacks is doubtful. Lastly, based on the observation on the immune response and the course of viral infection, it was unclear that the above result was obtained by the immune response induced by the vaccine. That is, immunological evidence which distinguish the case from the natural recovery (about 50%) was not enough to support the protective effect of the vaccine.
A DNA vaccine is superior to a protein vaccine in inducing cellular immune response. Since the antigen of DNA vaccine is expressed in host cells, it can induce humoral immunity with almost native conformation, and even simultaneously induce CD8+ T cell response, a kind of cellular immunity that a protein vaccine cannot induce, so that a DNA vaccine enhances the protective immunity to the maximum and easily induces Th1 immune response simply through the intramuscular injection (Pertmer, J. Virol., 1996, 70:6119). Unlike an inactivated vaccine or a killed vaccine, a DNA vaccine uses only a specific region of the virus as an antigen, causing fewer side effects. In addition, it is easy to store and convey a DNA vaccine, and the purification of the plasmid is also simple, comparing to other vaccines. The safety of a DNA vaccine was approved by Food and Drug Administration (FDA), USA, so that an AIDS DNA vaccine was allowed for clinical study in 1996. After the successful induction of immune response in small animal model, DNA immunization has been tried in many large animal models. Unfortunately, protective immunity against a highly pathogenic virus infection was not secured in large animal models. In experiments with HCV DNA vaccine in chimpanzees, the antibody and cellular immunity induced by DNA vaccine alone were so weak (Forns, Hepatology, 2000, 32:618) that another type of vaccine capable of inducing a strong cellular immunity was required.
Even though the limitation of a DNA vaccine has been widely known, the merit of the vaccine that can prime delicate immune response and Th1 immunity encouraged the study to overcome the limitation by combining with other boosting method. Previous reports suggest that the effect of the vaccine is greatly enhanced when boosted with a recombinant protein or an attenuated recombinant virus after priming with DNA. Such attempts were successful in small animals by inducing protective immunity against challenges (Song, J. Virol., 2000, 74:2920; Hanke, 1998, Vaccine, 16: 439-45; Sedegah, 1998, Proc. Natl. Acad. Sci. USA, 95: 7648-53; Schneider, 1998, Nat Med, 4: 397-402), and so was in Primates (Kent, 1998, J Virol, 72: 10180-8; Robinson, 1999, Nat Med, 5: 526-34; Amara, 2001, Science, 292: 69-74). Yet, there has been no report on the protective effect of DNA prime and adenovirus boosting regimen against hepatitis C virus infection.
Adenovirus has been proved safe and widely used as a vector for gene therapy. It was also proved to be very useful as a vaccine since it could induce strong humoral, cellular immune responses in various animal models (Natuk, Proc. Natl. Acad. Sci. USA, 1992, 89(16): 7777; Bruce, J. Gen. Virol., 1999, 80:2621). An earlier report demonstrated that the induced immune response was maintained for a long time after single injection of a replication-defective adenovirus (Juillard, Eur. J. Immunol., 1995, 25:3467). The antibody response and cellular immune response were also induced in small animal models by immunization with recombinant adenovirus expressing HCV structural gene (Makimura, Vaccine, 1996, 14:28; Bruna-Romero, Hepatology, 1997, 25:470; Seong, Vaccine, 2001, 19:2955). However, the induced Th1 immunity including CTL response was not compared in parallel with a DNA vaccine. Under this circumstance, DNA priming and adenovirus boosting regimen was tested in monkey model and proved its potential as a next generation vaccine regimen (Sullivan, 2000, Nature, 408: 605-9; Shiver, 2002, Nature, 415: 331-5).
The present inventors developed a DNA vaccine which induces optimal level of cellular immune response to hepatitis C virus through antigen engineering and confirmed that optimal Th1 immune response was induced by DNA priming and adenovirus boosting regimen. Finally, the present inventors proved in chimpanzee study that the vaccine regimen of the invention could induce the protective immunity against hepatitis C virus infection.